This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vaccination with vaccinia virus induces a vigorous virus-specific CD8-positive T cell response that plays an important role in control of poxvirus infection. However, little is known about the specific viral proteins that are serve as targets of vaccinia-specific CD8-positive T cells, and identification of T cell epitopes in a genome that encodes over 200 open reading frames (ORFs) is a particularly challenging task. As one approach to the identification of immunodominant poxvirus proteins, we used an algorithm for the prediction of vaccinia peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 vaccinia ORFs, screened these peptides for recognition by T cells from vaccinated macaques and identified immunogenic vaccinia proteins. These macaques developed relatively strong responses against multiple orthopoxvirus antigens: vaccinated macaques recognized 29 peptides from 26 different ORFs with 4 peptides recognized by all 3 macaques and 7 peptides recognized by 2 of 3 macaques. Eight epitopes did not contain the canonical P3 proline, suggesting that this residue is not required for in vivo presentation by Mamu-A*01. Comparison with other orthopoxvirus sequences revealed that these epitopes were highly conserved and present in vaccinia, variola, and monkeypox. These results suggest that the virus-specific CD8-positive T cell response is broadly directed against multiple vaccinia proteins and that a subset of these T cell epitopes are highly conserved among orthopoxviruses. AIDS related.